Factor VIII (FVIII) is a protein found in blood plasma which acts as a cofactor in the cascade of reactions leading to blood coagulation. A deficiency in the amount of FVIII activity in the blood results in the clotting disorder known as hemophilia A, which is primarily a congenital condition but can also be acquired in rare cases. Hemophilia A is currently treated with therapeutic preparations of FVIII derived from human plasma or manufactured using recombinant DNA technology. FVIII can be administered in response to a bleeding episode (on-demand therapy) and/or at frequent, regular intervals to prevent uncontrolled bleeding (prophylaxis).
Up to 30% of patients with severe hemophilia A (FVIII activity <1%) develop inhibitory antibodies to FVIII as a consequence of treatment with therapeutic preparations of FVIII (Lusher et al., J Thromb Haemost; 2:574-583 (2004); Scharrer et al., Haemophilia; 5:145-154 (1999)). Frequently, the inhibitors are persistent and of sufficiently high titer that infusion of FVIII concentrates is ineffective for controlling bleeding episodes. Inhibitor formation therefore represents a major obstacle in treating patients with hemophilia A. In patients with high titer inhibitors, acute bleeding can sometimes be controlled by infusion of bypass clotting factors, including activated prothrombin complex concentrates and recombinant human Factor VIIa. Bypass factors are considerably more expensive than standard FVIII concentrates, and their use in long-term prophylaxis regimens is limited due to their thrombogenic potential and unreliable hemostatic profile (Hay et al., Br J Haematol; 133:591-605 (2006); Paisley et al., Haemophilia; 9:405-417 (2003)). As a result, patients with high titer inhibitors have a markedly reduced quality of life due to frequent joint bleeds and the early progression of arthropathies (Morfini et al., Haemophilia; 13:606-612 (2007)).
At present, the only effective clinical protocols for immune tolerance induction (ITI) to FVIII involve daily administration of FVIII concentrate over the course of many months to 2 years. Administration of large quantities of soluble antigens has long been known to induce non-responsiveness to subsequent immunological challenge, but the high doses required and the inconsistency of tolerance induction make antigen administration alone impractical for most therapeutic agents. For example, ITI using FVIII is expensive, with a cost of approximately $1 million per treated patient (Colowick et al., Blood; 96:1698-1702 (2000)), and the mechanism by which it works is unknown. ITI is effective 60-80% of the time (Dimichele, J Thromb Haemost; 5 Suppl 1:143-150 (2007)) and its high costs can be offset by projected reductions in patient mortality and total lifetime treatment costs (Colowick et al., Blood; 96:1698-1702 (2000)). However, the morbidity and mortality suffered by patients with high titer FVIII inhibitors, the significant expense and high (20-40%) failure rate of current ITI protocols, and the extreme financial cost and limited effectiveness of alternative hemostatic agents all underscore the need to develop quicker, more reliable, and less expensive methods for tolerance induction.
Studies have shown that autologous cells undergoing apoptosis in the normal course of tissue turnover (under steady state conditions) are processed by unactivated dendritic cells (DCs) which phagocytose the apoptotic cells (ACs) and present AC antigens in the context of MHC Class I and II molecules to regulatory T cells capable of mediating antigen specific tolerance (Peng et al., J Autoimmun 29:303-309, (2007)). Regulatory T cells act to generate antigen specific tolerance through a variety of mechanisms including down regulation of DC activation, competition with effector T cells for access to antigen on DCs, and direct suppression of effector T cell proliferation by cytokine mediated inhibition or through Fas-Fas ligand cytolytic deletion (Misra et al., J Immunol; 172:4676-4680 (2004); Zhang et al.; Nat Med; 6:782-789 (2000); Vigoroux et al.; Blood; 104:26-33 (2004); Rutella et al.; Immunol Lett; 94:11-26 (2004)) Further research has shown that the immune response to a foreign antigen can also be suppressed by delivering it systemically in association with syngeneic ACs (Liu et al., J Exp Med; 196: 1091-1097 (2002); Ferguson et al., J Immunol; 168:5589-5595 (2002)). In contrast, immunity rather than tolerance was stimulated against AC-associated antigens when ACs were processed by DCs in the presence of an activation signal provided by an agonistic anti-CD40 antibody (Liu et al. (2002)). Thus, antigen presentation by DCs can lead to either immune priming or tolerance induction; it is the activation state of the DCs that process and present the antigen that determines the fate of the subsequent immune response (IR) (Moser, Immunity; 19:5-8 (2003); Probst et al., Immunity; 18:713-720 (2003)).
DCs exist in multiple phenotypically distinct subpopulations. Tolerogenic DCs are unactivated, expressing low levels of the T cell costimulatory molecules CD80 and CD86 and MHC Class II. These DCs also do not secrete pro-inflammatory cytokines such as IL-12, TNF-α, and IL1-β and therefore lack the ability to stimulate effector T cells. In contrast, tolerogenic DCs generate anti-inflammatory cytokines such as IL-10 that prevent immune priming of effector T cells and provide autocrine signals serving to keep DCs relatively resistant to activation (Sauter et al., J Exp Med; 191:423-434 (2000); Steinman et al., Annu Rev Immunol; 21:685-711 (2003); Stuart et al., J Immunol; 168:1627-1635 (2002)). Thus, the processing of autologous ACs by unactivated tolerogenic DCs does not produce an immune priming response, allowing for the induction of tolerance to AC-associated antigens.
ACs are more than just passive participants in this process. After phagocytosing ACs, immature DCs become more resistant to maturation and activation, in part due to blockade of NF-κB activation (Sen et al., Blood; 109: 653-660 (2007); Stuart et al., J Immunol; 168:1627-1635 (2002)). Furthermore, phagocytes that ingest ACs show decreased production of proinflammatory cytokines including IL-12, TNF-α, and IL1-β along with increased generation of the anti-inflammatory cytokine IL-10 (Kim et al., Immunity; 21:643-653 (2004); Voll et al., Nature; 390:350-351 (1997)). ACs also release their own anti-inflammatory signals, such as transforming growth factor (TGF)-β (Chen et al., Immunity; 14:715-725 (2001)), which may inhibit effector T cells at the sites of antigen processing and presentation.
The ability to induce peripheral tolerance by presenting antigens in association with apoptotic cells suggests that such cells might be useful vehicles for administering FVIII to prevent generation of high titer anti-FVIII inhibitory antibodies. To this end, a syngeneic fibroblast cell line was developed from an FVIII knockout (KO) mouse, and the cells were transduced with a vector expressing a human FVIII construct (Su et al., Blood (ASH Annual Meeting Abstracts); 106: 216 (2005); Su et al., Blood (ASH Annual Meeting Abstracts); 108: 768 (2006)). The transduced cells were induced to undergo apoptosis and then administered to FVIII KO mice prior to immunization with 4 doses of recombinant human FVIII. Mice that received apoptotic cells expressing the FVIII construct had reduced inhibitor titers and T cell responses compared to controls (Su et al. (2005); Su et al. (2006)). However, mice that were re-challenged with additional doses of rhFVIII 4 months after the initial immunization all showed significant boosting of the titers of inhibitory antibodies to FVIII (Su et al. (2006)). Thus the initial suppression of the immune response to FVIII by the vector modified apoptotic fibroblasts did not result in durable tolerance. Furthermore, the findings of Su et al. were limited to subjects that were immunologically naive to FVIII. Thus, the studies did not address whether the methods might be applicable to subjects having preformed immune responses to FVIII, such as the large population of hemophilia A patients who have developed high titer inhibitors to FVIII as a consequence of therapeutic infusions of FVIII concentrates.
Accordingly, there is a need in the art for safe, effective, and low cost treatments for hemophilia A patients with inhibitors to FVIII, as well as hemophilia A patients that are immunologically naive to FVIII.